Biocompatible polymer and magnetic nanoparticle with biocompatibility

ABSTRACT

The invention discloses a biocompatible polymer for covalently modifying magnetic nanoparticles. The biocompatible polymer may be coupled to a targeting agent and/or a fluorescent dye. The invention also discloses a magnetic nanoparticle with biocompatibilities comprising the biocompatible polymer.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a biocompatible polymer and in particular to abiocompatible polymer for covalently modifying magnetic nanoparticles.

2. Description of the Related Art

Magnetic resonance imaging (MRI) is an appealing noninvasive approachfor early cancer diagnostics and therapeutics. MRI utilizes radiofrequency pulses and magnetic field gradients applied to a subject in astrong field to produce images. MRI is capable of showing severaldifferent characteristics of tissues. The level of tissue magnetizationat specific signal recording periods during the MR imaging cyclegenerally determines the brightness of a particular tissue in the MRIimages. Contrast is produced when tissues do not have the same level ofmagnetization.

While the imaging capabilities of MRIs have revolutionized imagingtechnology, the resolution is limited to the elucidations of lesionswithin the body on the order of 1 mm. This limitation has led to thedevelopment of contrast enhancement agents. Because of thesuperparamagnetic property, iron oxide nanoparticles have been foundeffective as contrast enhancement agents for MRIs. The magneticnanoparticle can be modified with a biocompatible polymer to prolong theparticle circulation time in blood and reduce immunogenicity.Furthermore, the magnetic nanoparticle can be modified with afluorescent dye and a specific targeting agent to provide fluorescentproperties and specific targeting functions.

U.S. Patent Publication No. 20070148095 discloses a multi-modalitycontrast agent with specificity for both magnetic and optical imaging.The multi-modality contrast agent includes a magnetic nanoparticle, abiocompatible polymer chemically modifying the magnetic nanoparticle, afluorescent dye coupled to the biocompatible polymer, and a specifictargeting agent coupled to the biocompatible polymer. The biocompatiblepolymers include polyethylene glycol (PEG), polylactic acid (PLA),PLA-PEG, poly(glycolic acid) (PGA), poly(ε-caprolactone) (PCL),poly(methyl methacrylate) (PMMA), and the like.

U.S. Patent Publication No. 20070148095 discloses a silane compound formodifying magnetic nanoparticle and a method for using the nanoparticleto detect and treat tissues of interest.

Commercially available MRI contrast enhancement agents include Feridex®(dextran-coated iron oxide) and Resovist® (carboxydextran-coated ironoxide).

BRIEF SUMMARY OF THE INVENTION

In one aspect, the invention provides a biocompatible polymer of formula(I),

wherein R₁ is alkyl, aryl, carboxyl, or amino, R₂ is alkyl or aryl, n isan integer from 5 to 1000, and m is an integer from 1 to 10.

In another aspect, the invention provides a magnetic nanoparticle withbiocompatibility, comprising a magnetic nanoparticle and a biocompatiblepolymer of formula (II) covalently coupled to the magnetic nanoparticle,

wherein R₁ is alkyl, aryl, carboxyl, or amino, n is an integer from 5 to1000, and m is an integer from 1 to 10.

A detailed description is given in the following embodiments withreference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention can be more fully understood by reading the subsequentdetailed description and examples with references made to theaccompanying drawings, wherein:

FIG. 1 is a schematic drawing showing the synthesis of the biocompatiblepolymer of the invention; and

FIG. 2 is a schematic drawing showing a magnetic nanoparticle modifiedwith the biocompatible polymer of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The following description is of the best-contemplated mode of carryingout the invention. This description is made for the purpose ofillustrating the general principles of the invention and should not betaken in a limiting sense. The scope of the invention is best determinedby reference to the appended claims.

The biocompatible polymer of the invention is represented by generalformula (I),

wherein R₁ is alkyl, aryl, carboxyl, or amino, R₂ is alkyl or aryl, n isan integer from 5 to 1000, and m is an integer from 1 to 10. FIG. 1 is aschematic drawing showing the synthesis of the biocompatible polymer ofthe invention, wherein R1, R2, n, and m have the same meaning asdescribed above. As shown in FIG. 1, the synthetic scheme involvesconverting the hydroxyl end group of polyethylene glycol (PEG) to acarboxyl group by using a succinic anhydride compound, and coupling asilane group to the PEG. Suitable alkyl groups for R₁ and R₂ includeC₁-C₂₀ straight chain or branched alkyl groups. In one embodiment, eachof R₁ and R₂ independently, is a C₁-C₆ straight chain or branched alkylsuch as methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl,tert-butyl, n-pentyl, isopentyl, tert-pentyl, n-hexyl, and isohexyl.Suitable aryl groups for R₁ and R₂ include C₆-C₁₂ substituted orunsubstituted aryl groups such as phenyl, biphenyl, and naphthyl, andexamples of substituents thereof include hydroxyl, haloalkyl, alkoxyl,cyano, nitro, amino, or alkylamino. The number of methylene units m ispreferably an integer from 1 to 10. The number of oxyethylene units n ispreferably an integer from 5 to 1000, equivalent to a molecular weightof about 200-50000 g/mole of the PEG. In one embodiment, m is about 3,and n is about 15.

The biocompatible polymer synthesized in FIG. 1 is useful in that it canchemically modify the surface of the iron oxide nanoparticle to increasebiocompatibility. In addition, the biocompatible polymer is useful inthat it can label particles (e.g., nanoparticles, magnetic particles,magnetic nanoparticles, superparamagnetic particles), to render theparticles to be further reactive toward one or more targeting,fluorescent, therapeutic, or diagnostic agents.

The invention also provides a magnetic nanoparticle withbiocompatibility, comprising a magnetic nanoparticle; a biocompatiblepolymer of formula (II) covalently coupled to the magnetic nanoparticle,

wherein R₁ is alkyl, aryl, carboxyl, or amino, n is an integer from 5 to1000, and m is an integer from 1 to 10. FIG. 2 is a schematic drawingshowing a magnetic nanoparticle modified with the biocompatible polymerof the invention. The magnetic nanoparticle is preferably made of atleast one of Fe, Co, Ni, and oxides thereof. It will be appreciated thatthe nanoparticle can be made of any single or composite magneticmaterial, although superparamagnetic materials are particularlypreferred. After the biocompatible polymer is chemically bonded to themagnetic nanoparticle, the terminal groups R₁ are transformed intoactive functional groups such as carboxyl or amino groups to allowcoupling with fluorescent dye and/or specific targeting agents. However,R₁ is not alkyl or aryl, since alkyl or aryl are not capable of couplingwith targeting agents or fluorescent dye. In one embodiment, R₁ is acarboxyl group. The number of methylene units m is preferably an integerfrom 1 to 10. The number of oxyethylene units n is preferably an integerfrom 5 to 1000. In one embodiment, m is about 3, and n is about 15. Thebiocompatible polymer is preferably coated on the entire surface of themagnetic nanoparticle to form a core-shell structure. More preferably,the biocompatible polymer forms a monolayer coating on the magneticnanoparticle.

Experimental results indicate that the biocompatible polymer of theinvention may increase the r2 value of the magnetic nanoparticle toabout 2 times that of commercial contrast agents, Feridex® andResovist®. Accordingly, the magnetic nanoparticle may provide greatercontrast enhancement when being used as an MRI contrast agent.

The targeting agent is preferably coupled to the biocompatible polymervia covalent bonds. Commonly used targeting agents include an antibody,a protein, a peptide, an enzyme, a carbohydrate, a glycoprotein, anucleotide, and a lipid. The magnetic nanoparticle may have a diameterof about 3-500 nm after coupling with the targeting agent. Those skilledin the art can attach any suitable targeting agents on the nanoparticleto give specificity thereto. For example, folic acid can be used tospecify breast cancer cells with a folate receptor. The structure of thefolic acid allows coupling with an amine-terminated orcarboxy-terminated biocompatible polymer. For example, the folic acidallows coupling with the amine-terminated biocompatible polymer byforming a —CONH— linkage.

A fluorescent dye may be further coupled to the magnetic nanoparticle toprovide an optical signal for optical imaging techniques such as NIRimaging, thus allowing real-time monitoring of foci by different imagingtechniques. Preferably, the fluorescent dye is coupled to thebiocompatible polymer via covalent bonds. Suitable fluorescent dyesinclude organic or inorganic dyes and organometallic complexes. Theexcitation and emission wavelengths of the fluorescent dye may beultraviolet (UV), near-infrared (NIR), or visible (VIS) light. Themagnetic nanoparticle coupled with the targeting agent and fluorescentdye preferably has a diameter of about 15-200 nm.

Without intending to limit the present invention in any manner, thepresent invention will be further illustrated by the following examples.

Example 1 Nanoparticle Preparation

11.6 g (0.058 mole) of FeCl₂. 4H₂O, 11.6 g (0.096 mole) of FeCl₃.6H₂Oand 400 ml of deionized water were stirred in a three-necked flask at300 rpm at 25° C. 170 ml of a 2.5N NaOH solution was added to the flaskat a rate of 47 μl/sec. When a pH value of 11-12 was measured after theaddition of the 2.5N NaOH solution, 20 ml of oleic acid was added andstirred for 30 minutes. Thereafter, a 6N HCl solution was slowly addedto adjust the pH value to about 1, thus precipitating oleic acidencapsulated-iron oxide particles. The precipitates were collected,washed with deionized water for 4-5 times to remove excess oleic acid,and dried.

Example 2 Synthesis of Biocompatible Polymer mPEG-silane

300 g (0.4 mole) of methoxy-PEG (mPEG, molecular weight: 750) and 600 mlof N-methyl-2-pyrrolidone were placed in a 1000 ml round bottom flaskunder vacuum (20 Ton) for more than 2 hours. 48 g (0.48 mole) ofsuccinic anhydride and 19.5 g (0.159 mole) of 4-dimethylamino-pyridine(DMAP) were added for reaction at 30° C. for two days.

36 ml (0.48 mole) of thionyl chloride was added at a rate of 1 ml/minand the mixture was stirred for 2-3 hours. Thereafter, 133.8 ml (0.96mole) of triethylamine was added at a rate of 1 ml/min. After cooled toroom temperature, the mixture was filtered to remove precipitates. 94.5ml (0.4 mole) of 3-aminopropyl triethoxysilane was added for reactionfor at least 8 hours.

The reaction mixture was added to 9 L of isopropyl ether forre-precipitation, and the precipitates were collected, re-dissolved in500 ml of toluene, and centrifuged at 5000 rpm for 5 minutes to collecta supernatant. The supernatant was again, added to 9 L of isopropylether for re-precipitation. Brown oily liquid was collected and driedunder vacuum to obtain the biocompatible polymer, mPEG-silane.

Example 3 Synthesis of Biocompatible Polymer COOH-PEG-silane

300 g (0.4 mole) of PEG (molecular weight: 750) and 600 ml ofN-methyl-2-pyrrolidone were placed in a 1000 ml round bottom flask undervacuum (20 Ton) for more than 2 hours. 96 g (0.96 mole) of succinicanhydride and 39 g (0.318 mole) of 4-dimethylamino-pyridine (DMAP) wereadded for reaction at 30° C. for two days, thus obtainingdicarboxy-terminated PEG (COOH-PEG).

36 ml (0.48 mole) of thionyl chloride was added at a rate of 1 ml/minand stirred for 2-3 hours. Thereafter, 133.8 ml (0.96 mole) oftriethylamine was added at a rate of 1 ml/min. After cooled to roomtemperature, the mixture was filtered to remove precipitates. Then, 94.5ml (0.4 mole) of 3-aminopropyl triethoxysilane was added for reactionfor at least 8 hours.

The reaction mixture was added to 9 L of isopropyl ether forre-precipitation, and the precipitates were collected, re-dissolved in500 ml of toluene, and centrifuged at 5000 rpm for 5 minutes to collecta supernatant. The supernatant was again, added to 9 L of isopropylether for re-precipitation. Brown oily liquid was collected and driedunder vacuum, thus obtaining the biocompatible polymer, COOH-PEG-silane.

Example 4 Coupling with Biocompatible Polymer

250 g of mPEG-silane or COOH-PEG-silane was added to 1-1.2 L of atoluene solution containing 10 g of iron oxide of Example 1 and themixture was sonicated for 2-3 hours. After addition of 1.5 L ofdeionized water, the mixture was purified by an ultra-filtration deviceand concentrated to 100 ml to obtain iron oxide nanoparticles modifiedby a biocompatible polymer.

Example 5 Coupling with Targeting Agent

226 μl of folate solution (folate/dimethyl sulfoxide: 10 mg/ml) wasplaced in a 50 ml brownish round bottom flask. 5 ml of dimethylsulfoxide (DMSO) and 176.5 μl of dicyclohexyl carbodiimide solution(dicyclohexyl carbodiimide/DMSO: 5 mg/ml) was added to the solution andstirred for 1 hour. Thereafter, 98.5 μl of NHS solution(N-hydroxysuccinimide/DMSO: 5 mg/ml) was added and stirred for 1 hour.Then, 289 μl of ethylenediamine was added to give a solution A.

1 ml of the COOH-PEG-silane modified iron oxide nanoparticle of Example4 (4.48 mg/ml) and 10 ml of DMSO were placed in a 50 ml round bottomflask under vacuum for 1 hour. 176.5 μl of dicyclohexyl carbodiimidesolution (dicyclohexyl carbodiimide/DMSO: 5 mg/ml) was added to thesolution and stirred for 1 hour. Thereafter, 98.5 μl of NHS solution(N-hydroxysuccinimide/DMSO: 5 mg/ml) was added and stirred for 1 hour togive a solution B.

2895 μl (half-volume) of solution A was added to solution B and stirredfor 8 hours. The resulting solution was added into a dialysis membrane(Mw: 3000) and water was used for dialysis. Then, the solution wasconcentrated to 2 ml by an ultra-filtration device to obtain iron oxidenanoparticles coupled with a targeting agent.

Example 6 Coupling with Fluorescent Dye

1 ml of CypHer5E (NIR dye from Amersham Bioscience Co., 10⁻⁶ mole/ml)was mixed with 10⁻⁶ mole of ethylenediamine and stirred for 1 hour, thusgiving a solution C.

The iron oxide nanoparticles coupled with folate (2 mg/ml) of Example 5were dissolved in 10 ml of deionized water, followed by addition of 10⁻⁶mole of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). After themixture was stirred for one hour, 10⁻⁶ mole of N-hydroxysuccinimide(NHS) was added and stirred for another hour, thus giving a solution D.

Solution C was added to solution D and stirred for 8 hours. Theresulting solution was added into a dialysis membrane (Mw: 3000) andwater was used for dialysis. Then, the solution was concentrated to 2 mlby an ultra-filtration device to obtain iron oxide nanoparticles coupledwith a targeting agent and a fluorescent dye.

Example 7 Relaxivity Test

The modified iron oxide nanoparticles of Example 5 were compared for ther1 and r2 relaxivity with the product of U.S. Patent Publication No.2006/0216239 and commercial contrast agents, i.e., Feridex® andResovist®.

Iron oxide solutions of various concentrations (0.1, 0.2, 0.3, 0.4, 0.5mM) were prepared and measured for the T1 or T2 relaxation time by aMinispec mq 20 from the Bruker Corporation. A linear relationship wasestablished between the reciprocal of relaxation time as the ordinateaxis and the concentration of the solution as the abscissa axis. Theslope of the linear relationship was the r1 and r2 relaxivity.

As shown in Table 1, the r2 relaxivity of the modified iron oxidenanoparticles of the invention was about 2 times that of Feridex® andResovist®, and about 1.4 times that of the prior art product of U.S.Patent Publication No. 2006/0216239. Accordingly, the contrastenhancement was improved due to the higher r2 relaxivity.

TABLE 1 The US invention 2006/0216239 Resovist ® Feridex ® Diameter*8-12 nm 8-12 nm 4.2 nm 4.8-5.6 nm r2 (mM · 321.8 ± 2.3 229 164 160 s)⁻¹r1 (mM ·  33.4 ± 0.3 23.6 25.4 40 s)⁻¹ *The diameter was determined byTEM

While the invention has been described by way of example and in terms ofpreferred embodiment, it is to be understood that the invention is notlimited thereto. To the contrary, it is intended to cover variousmodifications and similar arrangements (as would be apparent to thoseskilled in the art). Therefore, the scope of the appended claims shouldbe accorded the broadest interpretation so as to encompass all suchmodifications and similar arrangements.

1. A biocompatible polymer of formula (I),

wherein R₁ is alkyl, aryl, carboxyl, or amino; R₂ is alkyl or aryl; n isan integer from 5 to 1000; and m is an integer from 1 to
 10. 2. Thebiocompatible polymer as claimed in claim 1, wherein each of R₁ and R₂independently is a C₁-C₂₀ straight chain or branched alkyl.
 3. Thebiocompatible polymer as claimed in claim 1, wherein each of R₁ and R₂independently is a C₆-C₁₂ substituted or unsubstituted aryl.
 4. Thebiocompatible polymer as claimed in claim 1, wherein R₁ is methyl. 5.The biocompatible polymer as claimed in claim 1, wherein R₂ is ethyl. 6.The biocompatible polymer as claimed in claim 1, wherein n is
 15. 7. Thebiocompatible polymer as claimed in claim 1, wherein m is
 3. 8. Amagnetic nanoparticle with bio compatibility, comprising: a magneticnanoparticle; a biocompatible polymer of formula (II) covalently coupledto the magnetic nanoparticle,

wherein R₁ is alkyl, aryl, carboxyl, or amino; n is an integer from 5 to1000; and m is an integer from 1 to
 10. 9. The magnetic nanoparticlewith biocompatibility as claimed in claim 8, wherein R₁ is carboxyl oramino.
 10. The magnetic nanoparticle with biocompatibility as claimed inclaim 8, wherein the magnetic nanoparticle is a superparamagneticnanoparticle.
 11. The magnetic nanoparticle with biocompatibility asclaimed in claim 8, wherein the magnetic nanoparticle comprises at leastone of Fe, Co, Ni, and oxides thereof.
 12. The magnetic nanoparticlewith biocompatibility as claimed in claim 9, wherein R₁ is coupled to aspecific targeting agent comprising an antibody, a protein, a peptide,an enzyme, a carbohydrate, a glycoprotein, a nucleotide or a lipid. 13.The magnetic nanoparticle with biocompatibility as claimed in claim 12,wherein the magnetic nanoparticle has a diameter of about 3-500 nm. 14.The magnetic nanoparticle with biocompatibility as claimed in claim 12,wherein the biocompatible polymer is coupled to a fluorescent dye. 15.The magnetic nanoparticle with biocompatibility as claimed in claim 14,wherein the magnetic nanoparticle containing the fluorescent dye and thespecific targeting agent has a diameter of about 15-200 nm.
 16. Themagnetic nanoparticle with biocompatibility as claimed in claim 14,wherein the fluorescent dye exhibits at least one of ultraviolet (UV),near-infrared (NIR), and visible (VIS) light excitation or emissionwavelength.
 17. The magnetic nanoparticle with biocompatibility asclaimed in claim 14, wherein the fluorescent dye comprises an organicdye, an inorganic dye, or an organometallic complex.
 18. The magneticnanoparticle with biocompatibility as claimed in claim 14, wherein thefluorescent dye and the specific targeting agent are coupled to thebiocompatible polymer by a covalent bond.
 19. The magnetic nanoparticlewith biocompatibility as claimed in claim 14, wherein the specifictargeting agent and the biocompatible polymer are coupled by a —CONH—linkage.
 20. The magnetic nanoparticle with biocompatibility as claimedin claim 8, wherein the biocompatible polymer is coated on the magneticnanoparticle to form a core/shell structure.
 21. The magneticnanoparticle with biocompatibility as claimed in claim 20, wherein thebiocompatible polymer forms a monolayer coating on the magneticnanoparticle.